In response to concerns that bioterrorists may release smallpox virus in the United States, the government has implemented a multi-stage vaccination program. However, the observation of cardiac adverse events following vaccination has resulted in the Centers for Disease Control recommending that people at risk for heart disease should be excluded from the vaccination program. Notably, myocarditis has been diagnosed in 18 recent smallpox vaccinees in the USA. Despite this recent temporal association between myocarditis and smallpox vaccination as well as a similar historic association, there have been no studies of the effects of poxviruses on cardiac cells, or the development of any animal models to study poxvirus-induced myocarditis. For other viruses, myocarditis can reflect a direct cytopathogenic effect of the virus on cardiac myocytes, or can be mediated by immune cells, cytokines, or other soluble factors. Poxviruses can be directly cytopathogenic, but also encode a number of modulators of cytokine responses. Cowpox virus contains the full complement of known orthopoxvirus accessory genes that affect immune responses, while DryVax (the current vaccine strain of vaccinia virus) encodes fewer accessory genes, and modified vaccinia Ankara virus (MVA, which is replication-defective in human cells) contains even fewer accessory genes. Our preliminary data demonstrate that cowpox virus and DryVax replicate in murine primary cardiac myocyte cultures (PCMCs) whereas MVA does not. Moreover, cowpox, DryVax, and MVA are each cytopathogenic to these cultures, though the type of cytopathogenicity is virus strain-specific. We hypothesize that poxviruses have virus strain-specific adverse effects on cardiac cells, either directly or indirectly, resulting in damage to cardiac myocytes and the heart. In Specific Aim 1, we will determine the effect of these three poxviruses (cowpox, DryVax, and MVA) on murine primary cardiac myocyte cultures (PCMCs) and control cultures, analyzing viral infection and gene expression, and induction of cytopathogenicity. In Specific Aim 2, we will determine the association between cytokines and poxvirus infection on cytopathogenicity to PCMCs and control cultures. In Specific Aim 3, we will determine whether these poxviruses induce myocarditis in a variety of mouse strains. Studies proposed in this R21 application will provide the foundation for future in-depth studies with the goal of improving current and future smallpox vaccines.